Calcium entry into myocytes drives contraction of the embryonic heart. To prepare for the next contraction, myocytes must extrude calcium from intracellular space via the Na⁺/Ca²⁺exchanger (NCX1) or sequester it into the sarcoplasmic reticulum, via the sarcoplasmic reticulum Ca²⁺-ATPase2 (SERCA2). In mammals, defective calcium extrusion correlates with increased intracellular calcium levels and may be relevant to heart failure and sarcoplasmic dysfunction in adults. We report here that mutation of the cardiac-specific NCX1 (NCX1h) gene causes embryonic lethal cardiac arrhythmia in zebrafishtremblor(tre) embryos. Thetreventricle is nearly silent, whereas the atrium manifests a variety of arrhythmias including fibrillation. Calcium extrusion defects intremutants correlate with severe disruptions in sarcomere assembly, whereas mutations in the L-type calcium channel that abort calcium entry do not produce this phenotype. Knockdown ofSERCA2activity by morpholino-mediated translational inhibition or pharmacological inhibition causes embryonic lethality due to defects in cardiac contractility and morphology but, in contrast totremutation, does not produce arrhythmia. Analysis of intracellular calcium levels indicates that homozygoustreembryos develop calcium overload, which may contribute to the degeneration of cardiac function in this mutant. Thus, the inhibition of NCX1h versus SERCA2 activity differentially affects the pathophysiology of rhythm in the developing heart and suggests that relative levels of NCX1 and SERCA2 function are essential for normal development.

Background: L‐type calcium channels (LTCC) regulate calcium entry into cardiomyocytes. CACNB2 (β2) LTCC auxiliary subunits traffic the pore‐forming CACNA subunit to the membrane and modulate channel kinetics. β2 is a membrane associated guanylate kinase (MAGUK) protein. A major role of MAGUK proteins is to scaffold cellular junctions and multiprotein complexes. Results: To investigate developmental functions for β2.1, we depleted it in zebrafish using morpholinos. β2.1‐depleted embryos developed compromised cardiac function by 48 hr postfertilization, which was ultimately lethal. β2.1 contractility defects were mimicked by pharmacological depression of LTCC, and rescued by LTCC stimulation, suggesting β2.1 phenotypes are at least in part LTCC‐dependent. Morphological studies indicated that β2.1 contributes to heart size by regulating the rate of ventricle cell proliferation, and by modulating the transition of outer curvature cells to an elongated cell shape during chamber ballooning. In addition, β2.1‐depleted cardiomyocytes failed to accumulate N‐cadherin at the membrane, and dissociated easily from neighboring myocytes under stress. Conclusions: Hence, we propose that β2.1 may also function in the heart as a MAGUK scaffolding unit to maintain N‐cadherin‐based adherens junctions and heart tube integrity. Developmental Dynamics 241:648–662, 2012. © 2012 Wiley Periodicals Inc.

Background: T‐box genes constitute a large family of transcriptional regulators involved in developmental patterning. Homozygous mutation of tbx5 leads to embryonic lethal cardiac phenotypes and forelimb malformations in vertebrate models. Haploinsufficiency of tbx5 results in Holt‐Oram syndrome, a human congenital disease characterized by cardiac and forelimb defects. Homozygous mutation of zebrafish tbx5a leads to lethal defects in cardiac looping morphogenesis, blocks pectoral fin initiation, and impairs outgrowth. Recently, a second zebrafish tbx5 gene was described, termed tbx5b. Results: Our phylogenetic analyses confirm tbx5b as a paralog that likely arose in the teleost‐specific whole genome duplication ∼270 MYA. Using morpholino depletion studies, we find that tbx5b is required in the heart for embryonic survival, and influences the timing and morphogenesis of pectoral fin development. Because tbx5a hypomorphic mutations are embryonic lethal, tbx5a and tbx5b functions in the heart must not be completely redundant. Consistent with this hypothesis, simultaneous depletion of both tbx5 paralogs did not lead to more severe phenotypes, and injection of wild‐type mRNA from one tbx5 paralog was not sufficient to cross‐rescue phenotypes of the paralogous gene. Conclusions: Collectively, these data indicate that, despite similar spatio‐temporal expression patterns, tbx5a and tbx5b have independent functions in heart and fin development. Developmental Dynamics 242:475–492, 2013.

Switchgrass (Panicum virgatum), a robust perennial C4-type grass, has been evaluated and designated as a model bioenergy crop by the U.S. DOE and USDA. Conventional breeding of switchgrass biomass is difficult because it displays self-incompatible hindrance. Therefore, direct genetic modifications of switchgrass have been considered the more effective approach to tailor switchgrass with traits of interest. Successful transformations have demonstrated increased biomass yields, reduction in the recalcitrance of cell walls and enhanced saccharification efficiency. Several tissue culture protocols have been previously described to produce transgenic switchgrass lines using different nutrient-based media, co-cultivation approaches, and antibiotic strengths for selection. After evaluating the published protocols, we consolidated these approaches and optimized the process to develop a more efficient protocol for producing transgenic switchgrass. First, seed sterilization was optimized, which led to a 20% increase in yield of induced calluses. Second, we have selected a N6 macronutrient/B5 micronutrient (NB)-based medium for callus induction from mature seeds of the Alamo cultivar, and chose a Murashige and Skoog-based medium to regenerate both Type I and Type II calluses. Third, Agrobacterium-mediated transformation was adopted that resulted in 50–100% positive regenerated transformants after three rounds (2 weeks/round) of selection with antibiotic. Genomic DNA PCR, RT-PCR, Southern blot, visualization of the red fluorescent protein and histochemical β-glucuronidase (GUS) staining were conducted to confirm the positive switchgrass transformants. The optimized methods developed here provide an improved strategy to promote the production and selection of callus and generation of transgenic switchgrass lines. The process for switchgrass transformation has been evaluated and consolidated to devise an improved approach for transgenic switchgrass production. With the optimization of seed sterilization, callus induction, and regeneration steps, a reliable and effective protocol is established to facilitate switchgrass engineering.

Cardiomyocyte contraction is initiated by influx of extracellular calcium through voltage-gated calcium channels. These oligomeric channels utilize auxiliary β subunits to chaperone the pore-forming α subunit to the plasma membrane, and to modulate channel electrophysiology [ 1 ]. Several β subunit family members are detected by RT-PCR in the embryonic heart. Null mutations in mouse β2, but not in the other three β family members, are embryonic lethal at E10.5 due to defects in cardiac contractility [ 2 ]. However, a drawback of the mouse model is that embryonic heart rhythm is difficult to study in live embryos due to their intra-uterine development. Moreover, phenotypes may be obscured by secondary effects of hypoxia. As a first step towards developing a model for contributions of β subunits to the onset of embryonic heart rhythm, we characterized the structure and expression of β2 subunits in zebrafish and other teleosts. Cloning of two zebrafish β2 subunit genes (β2.1 and β2.2) indicated they are membrane-associated guanylate kinase (MAGUK)-family genes. Zebrafish β2 genes show high conservation with mammals within the SH3 and guanylate kinase domains that comprise the "core" of MAGUK proteins, but β2.2 is much more divergent in sequence than β2.1. Alternative splicing occurs at the N-terminus and within the internal HOOK domain. In both β2 genes, alternative short ATG-containing first exons are separated by some of the largest introns in the genome, suggesting that individual transcript variants could be subject to independent cis-regulatory control. In the Tetraodon nigrovidis and Fugu rubripes genomes, we identified single β2 subunit gene loci. Comparative analysis of the teleost and human β2 loci indicates that the short 5' exon sequences are highly conserved. A subset of 5' exons appear to be unique to teleost genomes, while others are shared with mammals. Alternative splicing is temporally and spatially regulated in embryo and adult. Moreover, a different subset of spliced β2 transcript variants is detected in the embryonic heart compared to the adult. These studies refine our understanding of β2 subunit diversity arising from alternative splicing, and provide the groundwork for functional analysis of β2 subunit diversity in the embryonic heart.

The vertebrate embryonic heart initially forms with two chambers, a ventricle and an atrium, separated by the atrioventricular junction. Localized genetic and biomechanical information guides the development of valves, which function to ensure unidirectional blood flow. If the valve development process goes awry, pathology associated with congenital valve defects can ensue. Congenital valve defects (CVD) are estimated to affect 1–2% of the population and can often require a lifetime of treatment. Despite significant clinical interest, molecular genetic mechanisms that direct valve development remain incompletely elucidated. Cells in the developing valve must contend with a dynamic hemodynamic environment. A growing body of research supports the idea that cells in the valve are highly sensitive to biomechanical forces, which cue changes in gene expression required for normal development or for maintenance of the adult valve. This review will focus on mechanotransductive pathways involved in valve development across model species. We highlight current knowledge regarding how cells sense physical forces associated with blood flow and pressure in the forming heart, and summarize how these changes are transduced into genetic and developmental responses. Lastly, we provide perspectives on how altered biomechanical cues may lead to CVD pathogenesis.